Balshaw, seen by some as a surprise inclusion in Sir Clive Woodward's squad for the tour of New Zealand in June and July, hobbled off after only four minutes of the match, which Leeds went on to win 20-12.
The England full-back had already shown a couple of glimpses of class with two telling incursions into the Bath three-quarter line before he was forced to leave the field.
Lions boss Woodward will inevitably be concerned by yet another fitness setback for Balshaw ahead of the departure down under next month.
Balshaw's career over the past two years has been stalled by a succession of injuries, and his latest setback is unrelated to the knee problem he sustained during last month's Six Nations clash against Scotland.
First-half tries from Chris Bell and Andre Snyman, plus two penalties from Gordon Ross, secured the win for the Tykes, who led 20-9 at the break.
Fly-half Chris Malone kicked four penalties for Bath, who had Lions Danny Grewcock and Matt Stevens in their ranks, but it was not enough as their immaculate Twickenham cup final record was ended.